QC Measures

Low correlation!

Spearman correlation across replicates is low using a barcode threshold per oligo of 5! Spearman correlation of DNA is 0.29, RNA is 0.58 and DNA/RNA ratio is 0.52. Minimum allowed values are 0.85, 0.95, and 0.75.

Oligo Coorelatioin

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 1567 oligos with a minimum number of 5 barcodes. Second tab shows all 2016 oligos that have assigned barcodes.

Condition	A	B	#Oligos A	#Oligos B	#Oligos Joined	DNA spearman	RNA spearman	Ratio spearman	DNA log2 pearson	RNA log2 pearson	Ratio log2 pearson
HepG2	1	2	1662	1684	1564	0.33	0.60	0.53	0.32	0.70	0.62
HepG2	1	3	1662	1700	1556	0.29	0.61	0.55	0.28	0.71	0.63
HepG2	2	3	1684	1700	1582	0.31	0.58	0.52	0.31	0.68	0.62

Condition	A	B	#Oligos A	#Oligos B	#Oligos Joined	DNA spearman	RNA spearman	Ratio spearman	DNA log2 pearson	RNA log2 pearson	Ratio log2 pearson
HepG2	1	2	2031	2028	2016	0.28	0.53	0.45	0.27	0.61	0.51
HepG2	1	3	2031	2030	2018	0.27	0.53	0.46	0.26	0.59	0.51
HepG2	2	3	2028	2030	2016	0.28	0.51	0.43	0.28	0.58	0.50

Experiment statistic

The total number of oligos in this experiment is 2050 (defined by the assignment) with 58369 associated barcodes.

In average across replicates we see 2030 from 25281 average barcodes in the count data and around 71255 barcodes where not in the assignment.

condition	replicate	oligos dna/rna	matched barcodes	unknown barcodes dna/rna	% matched barcodes	total dna counts	total rna counts	avg dna counts per bc	avg rna counts per bc	avg dna/rna barcodes per oligo
HepG2	1	2031	25038	71955	25.81	212037	566550	2.19	5.84	12.33
HepG2	2	2028	25420	71141	26.33	201559	564222	2.09	5.84	12.53
HepG2	3	2030	25384	70668	26.43	197109	601951	2.05	6.27	12.50

Experiment	Barcodes	Counts	Average counts	Assigned barcodes	Assigned counts	Average assigned counts	Fraction assigned barcodes	Fraction assigned counts
HepG2_1_DNA	149172	294136	1.97	32993	75057	2.27	0.22	0.26
HepG2_2_DNA	153364	289183	1.89	34261	74150	2.16	0.22	0.26
HepG2_3_DNA	152734	285380	1.87	34281	73508	2.14	0.22	0.26
HepG2_1_RNA	190654	871820	4.57	38356	229135	5.97	0.20	0.26
HepG2_2_RNA	187952	878332	4.67	38144	233350	6.12	0.20	0.27
HepG2_3_RNA	192812	959790	4.98	38566	253313	6.57	0.20	0.26

Number of barcodes per oligo

Histogramm of number of barcodes per oligo. Median is blue, mean is red.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 1567 oligos with a minimum number of 5 barcodes. Second tab shows all 2016 oligos that have assigned barcodes.

--- title: "QC Measures" page-layout: full format: html: code-fold: true code-tools: true embed-resources: true anchor-sections: true smooth-scroll: true toc: true theme: light: flatly dark: darkly jupyter: python3 --- ```{python} #| echo: false # Imports from IPython.display import display, Image, Markdown import pandas as pd ``` ```{python} #| tags: [parameters] #| echo: false ``` ```{python} #| echo: false oligo_cor_merged_table_thresh = pd.read_csv(f"{workdir}/{statistics_all_oligo_cor_thresh}", sep='\t') oligo_cor_merged_table_all = pd.read_csv(f"{workdir}/{statistics_all_oligo_cor_all}", sep='\t') dna_spear = oligo_cor_merged_table_thresh['DNA_spearman'].min() rna_spear = oligo_cor_merged_table_thresh['RNA_spearman'].min() ratio_spear = oligo_cor_merged_table_thresh['Ratio_spearman'].min() ``` ```{python} #| echo: false threshold_dna_spear = 0.85 threshold_rna_spear = 0.95 threshold_ratio_spear = 0.75 important = dna_spear < threshold_dna_spear and rna_spear < threshold_rna_spear and ratio_spear < threshold_ratio_spear warning = dna_spear < threshold_dna_spear or rna_spear < threshold_rna_spear or ratio_spear < threshold_ratio_spear if important: display(Markdown(f''' ::: {"{.callout-important}"} ## Low correlation! Spearman correlation across replicates is low using a barcode threshold per oligo of {thresh}! Spearman correlation of DNA is {dna_spear:.2f}, RNA is {rna_spear:.2f} and DNA/RNA ratio is {ratio_spear:.2f}. Minimum allowed values are {threshold_dna_spear:.2f}, {threshold_rna_spear:.2f}, and {threshold_ratio_spear:.2f}. ::: ''')) elif warning: display(Markdown(f''' ::: {"{.callout-warning}"} ## Low correlation warning! At least one (not all) Spearman correlations across replicates is low using a barcode threshold per oligo of {thresh}! Spearman correlation of DNA is {dna_spear:.2f}, RNA is {rna_spear:.2f} and DNA/RNA ratio is {ratio_spear:.2f}. Minimum allowed values are {threshold_dna_spear:.2f}, {threshold_rna_spear:.2f}, and {threshold_ratio_spear:.2f}. ::: ''')) else: display(Markdown(f''' ::: {"{.callout-tip}"} ## Good Spearman correlation across replicates! The data has a good Spearman correlations across replicates using a barcode threshold per oligo of {thresh}! Spearman correlation of DNA is {dna_spear:.2f}, RNA is {rna_spear:.2f} and DNA/RNA ratio is {ratio_spear:.2f}. ::: ''')) ``` # Oligo Coorelatioin Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) `{python} "%d" % oligo_cor_merged_table_thresh['number_Oligos_Joined'].mean()` oligos with a minimum number of `{python} thresh` barcodes. Second tab shows all `{python} "%d" % oligo_cor_merged_table_all['number_Oligos_Joined'].mean()` oligos that have assigned barcodes. ```{python} #| echo: false def filter_table(df): df = df.round(2).drop(columns=['DNA_pearson','RNA_pearson', 'Ratio_pearson', 'fraction_Oligos_ReplicateA', 'fraction_Oligos_ReplicateB', 'NormSymmetry']) df.columns = df.columns.str.replace("_", " ", regex=False).str.replace("Replicate", " ", regex=False).str.replace("number ", "#", regex=False) return(df) ``` ::: {.panel-tabset} ## Threshold `{python} thresh` ```{python} #| echo: false oligo_cor_merged_table_thresh_filter = filter_table(oligo_cor_merged_table_thresh) oligo_cor_merged_table_thresh_filter.style.hide(axis="index").format( precision=2) ``` ## All ```{python} #| echo: false oligo_cor_merged_table_all_filter = filter_table(oligo_cor_merged_table_all) oligo_cor_merged_table_all_filter.style.hide(axis="index").format( precision=2) ``` ::: ::: {.panel-tabset} ## Threshold `{python} thresh` ::: {.panel-tabset} ### DNA ```{python} #| echo: false display(Image(f"{workdir}/{dna_oligo_coor_min_thre_plot }")) ``` ### RNA ```{python} #| echo: false display(Image(f"{workdir}/{rna_oligo_coor_min_thre_plot}")) ``` ### Ratio ```{python} #| echo: false display(Image(f"{workdir}/{ratio_oligo_min_thre_plot}")) ``` ::: ## All ::: {.panel-tabset} ### DNA ```{python} #| echo: false display(Image(f"{workdir}/{dna_oligo_coor_plot}")) ``` ### RNA ```{python} #| echo: false display(Image(f"{workdir}/{rna_oligo_coor_plot}")) ``` ### Ratio ```{python} #| echo: false display(Image(f"{workdir}/{ratio_oligo_coor_plot}")) ``` ::: ::: # Experiment statistic ```{python} #| echo: false df_merged = pd.read_csv(f"{workdir}/{statistics_all_merged}", sep='\t').round(2) n_oligos = df_merged['oligos design'].iloc[0] n_barcodes = df_merged['barcodes design'].iloc[0] avg_oligos = df_merged['oligos dna/rna'].mean() avg_matched_barcodes = df_merged['matched barcodes'].mean() avg_unknown_barcodes = df_merged['unknown barcodes dna/rna'].mean() ``` The total number of oligos in this experiment is `{python} f"{n_oligos}"` (defined by the assignment) with `{python} f"{n_barcodes}"` associated barcodes. In average across replicates we see `{python} f"{avg_oligos:.0f}"` from `{python} f"{avg_matched_barcodes:.0f}"` average barcodes in the count data and around `{python} f"{avg_unknown_barcodes:.0f}"` barcodes where not in the assignment. ::: {.panel-tabset} # DNA/RNA merged ```{python} #| echo: false df_merged.drop(columns=['oligos design','barcodes design', 'barcodes dna/rna'], inplace=True) df_merged.style.hide(axis="index").format( precision=2) ``` # DNA/RNA individual ```{python} #| echo: false df_single = pd.read_csv(f"{workdir}/{statistics_all_single}", sep='\t').round(2) df_single.style.hide(axis="index").format( precision=2) ``` ::: # Number of barcodes per oligo Histogramm of number of barcodes per oligo. Median is blue, mean is red. ::: {.panel-tabset} # DNA ```{python} #| echo: false display(Image(f"{workdir}/{counts_per_oligo_dna}")) ``` # RNA ```{python} #| echo: false display(Image(f"{workdir}/{counts_per_oligo_rna}")) ``` ::: # Activity Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise `NA`. First tab shows plots using (in average) `{python} "%d" % oligo_cor_merged_table_thresh['number_Oligos_Joined'].mean()` oligos with a minimum number of `{python} thresh` barcodes. Second tab shows all `{python} "%d" % oligo_cor_merged_table_all['number_Oligos_Joined'].mean()` oligos that have assigned barcodes. ::: {.panel-tabset} ## Threshold `{python} thresh` ```{python} #| echo: false display(Image(f"{workdir}/{activity_thresh}")) ``` ## All ```{python} #| echo: false display(Image(f"{workdir}/{activity_all}")) ``` :::